Molecular Genetics - Past Lecture Notes PDF

Suparada Khanaruksombat, Ph.D. Hoefnagels, M. l. (2018). Biology : the essentials (Third edition. ed.). 2 Suparada Khanaruksombat, Ph.D. 3  The history of molecular biology begins in the 20th century. Joachim Wilhelm Robert Feulgen...

Suparada Khanaruksombat, Ph.D. Hoefnagels, M. l. (2018). Biology : the essentials (Third edition. ed.). 2 Suparada Khanaruksombat, Ph.D. 3  The history of molecular biology begins in the 20th century. Joachim Wilhelm Robert Feulgen the Feulgen staining (2 September 1884 – 24 October 1955) - a German physician and chemist - In 1914 , developed a method for staining DNA (now known as the Feulgen stain) - Discovered plant and animal nuclear DNA ("thymonucleic acid") congeniality. http://www.odermatol.com/wp-content/uploads/fig_2j(27).jpg Suparada Khanaruksombat, Ph.D. http://www.jomfp.in/articles/2011/15/2/images/JOralMaxillofacPathol_2011_15_2_177_84488_u7.jpg 4  Frederick Griffith (1879–1941) British bacteriologist whose focus was the epidemiology and pathology of bacterial pneumonia. In January 1928, he reported what is now known as Griffith's Experiment, the first widely accepted demonstrations of bacterial transformation, whereby a bacterium distinctly changes its form and function. Suparada Khanaruksombat, Ph.D. https://en.wikipedia.org/wiki/File:Griffithm.jpg 5 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 6  https://lh3.googleusercontent.com/--nHKwogwKNE/Vqb1F5u0N-I/AAAAAAAARts/FXpULJvCkvk/s1600/04950361289206881.jpg In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered that the transforming molecule is DNA. “DNA is the transforming factor in bacteria” Suparada Khanaruksombat, Ph.D. Audesirk, T., Audesirk, G., & Byers, B. E. (2017). Biology : life on Earth (11th ed.). 7  In 1952, Alfred Hershey and Martha Chase confirmed that DNA is genetic material. Hershey shared the 1969 Nobel Prize in Physiology or Medicine with Max Delbrück and Salvador Luria for their “discoveries concerning the genetic structure of viruses”. Suparada Khanaruksombat, Ph.D. https://www.nobelprize.org/prizes/medicine/1969/summary/ 8  Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). Suparada Khanaruksombat, Ph.D. 9  In the late 1940s, several scientists began to investigate the structure of DNA. British researchers Maurice Wilkins and Rosalind Franklin used a technique called X-ray diffraction to study the DNA molecule. Wilkins and Franklin made several deductions from their experiments. First, a molecule of DNA is long and thin, with a uniform width of 2 nanometers (2 billionths of a meter). Second, DNA is helical, twisted like a spiral staircase. Third, DNA is a double helix; that is, two strands of nucleotides coil around one another. Fourth, DNA consists of repeating subunits. And fifth, the phosphates are probably on the outside of the helix. Suparada Khanaruksombat, Ph.D. Audesirk, T., Audesirk, G., & Byers, B. E. (2017). Biology : life on Earth (11th ed.). 10  James Watson and Francis Crick knew the general size and shape of a DNA molecule. Crick and Watson offered a detailed molecular model for the structure of DNA. Suparada Khanaruksombat, Ph.D. Hoefnagels, M. l. (2018). Biology : the essentials (Third edition. ed.). 11  In the 1940s, biochemist Erwin Chargaff analyzed the amounts of the four bases in DNA from organisms as diverse as bacteria, sea urchins, fish, and humans. Although the proportions of each base differ from species to species, for any given species, there are always equal amounts of adenine and thymine and equal amounts of guanine and cytosine called “Chargaff’s rule" https://upload.wikimedia.org/wikipedia/en/a/a1/Erwin_Chargaff.jpg Erwin Chargaff (1905 – 2002) Suparada Khanaruksombat, Ph.D. http://www.coxclasses.com/biology/bioch12/figure5.jpg 12  อัตราส่วนของเบสในโมเลกุล DNA สายคู่ข้อใดมีค่าเท่ากับ 1 a. C/T b. A/G c. A/T d. (A+C)/(G+T) e. (A+T)/(G+C) f. (A+G)/(C+T) Suparada Khanaruksombat, Ph.D. 13  Mason, K. A., & Raven, P. H. (2016). Biology (11th ed.). Suparada Khanaruksombat, Ph.D. 14  Figure 5.26 - Reece, J. B., & Campbell, N. A. (2011). Campbell biology (9th ed.) Suparada Khanaruksombat, Ph.D. 15  Mason, K. A., & Raven, P. H. (2016). Biology (11th ed.) Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.).. 16  Figure 3.16-3.17 - Mason, K. A., & Raven, P. H. (2016). Biology (Eleventh edition. ed.) Suparada Khanaruksombat, Ph.D. 17 Suparada Khanaruksombat, Ph.D. Audesirk, T., Audesirk, G., & Byers, B. E. (2017). Biology : life on Earth (11th ed.). 18  Figure 3.18 - Mason, K. A., & Raven, P. H. (2016). Biology (Eleventh edition. ed.) Suparada Khanaruksombat, Ph.D. 19 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.).. 20 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.).. 21  Rich phosphate groups (-) Rich arginine and lysine (+) Suparada Khanaruksombat, Ph.D. 22  DNA Replication Suparada Khanaruksombat, Ph.D. Mason, K. A., & Raven, P. H. (2016). Biology (11th ed.). 23  Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). Suparada Khanaruksombat, Ph.D. Mason, K. A., & Raven, P. H. (2016). Biology (11th ed.). 24  Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). Suparada Khanaruksombat, Ph.D. 25  The bacterium E. coli has a single chromosome of about 4.6 million nucleotide pairs. In a favorable environment, an E. coli cell can copy all of this DNA and divide to form two genetically identical daughter cells in considerably less than an hour. Each of your somatic cells has 46 DNA molecules in its nucleus, one long double-helical molecule per chromosome. In all, that represents about 6 billion nucleotide pairs, or over 1,000 times more DNA than is found in most bacterial cells. Suparada Khanaruksombat, Ph.D. 26 Suparada Khanaruksombat, Ph.D. https://www.veritasgenetics.com/our-thinking/whole-story 27  Mason, K. A., & Raven, P. H. (2016). Biology (11th ed.) Suparada Khanaruksombat, Ph.D. 28 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 29  Initiation Termination Elongation Suparada Khanaruksombat, Ph.D. 30  The replication of chromosomal DNA begins at particular sites called origins of replication, short stretches of DNA that have a specific sequence of nucleotides. At each end of a replication bubble is a replication fork, a Y-shaped region where the parental strands of DNA are being unwound. Helicases are enzymes that untwist the double helix at the replication forks, separating the two parental strands and making them available as template strands. Topoisomerase is an enzyme that helps relieve this strain by breaking, swiveling, and rejoining DNA strands. After the parental strands separate, single-strand binding proteins bind to the unpaired DNA strands, keeping them from re-pairing. Suparada Khanaruksombat, Ph.D. 31 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 32  The initial nucleotide chain that is produced during DNA synthesis is actually a short stretch of RNA, not DNA called a primer and is synthesized by the enzyme primase. The completed primer, generally 5–10 nucleotides long, is thus base-paired to the template strand. The new DNA strand will start from the 3′ end of the RNA primer. Enzymes called DNA polymerases catalyze the synthesis of new DNA by adding nucleotides to the 3′ end of a preexisting chain. In E. coli, there are several DNA polymerases, but two appear to play the major roles in DNA replication: DNA polymerase III (Pol III) and DNA polymerase I (Pol I). Suparada Khanaruksombat, Ph.D. 33  Each nucleotide to be added to a growing DNA strand consists of a sugar attached to a base and to three phosphate groups that called ATP. The only difference between the ATP of energy metabolism and dATP, the adenine nucleotide used to make DNA, is the sugar component, which is deoxyribose in the building block of DNA but ribose in ATP. Suparada Khanaruksombat, Ph.D. http://www.oezratty.net/wordpress/wp-content/WindowsLiveWriter/Les-dessous-techniques-du-squenage-du-gn_82A7/ATP-et-dATP.jpg 34 Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). Suparada Khanaruksombat, Ph.D. 35  DNA polymerases can add nucleotides only to the free 3′ end of a primer or growing DNA strand, never to the 5′ end. Thus, a new DNA strand can elongate only in the 5′ → 3′ direction. The DNA strand made by this mechanism is called the leading strand. To elongate the other new strand of DNA in the mandatory 5′ → 3′ direction, DNA pol III must work along the other template strand in the direction away from the replication fork. The DNA strand elongating in this direction is called the lagging strand. In contrast to the leading strand, which elongates continuously, the lagging strand is synthesized discontinuously, as a series of segments. These segments of the lagging strand are called Okazaki fragments. Reiji Okazaki (1930 – 1975) Suparada Khanaruksombat, Ph.D. 36 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 37 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 38 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology 39 Suparada Khanaruksombat, Ph.D. http://www.b4fa.org/wp-content/uploads/2012/07/Gene-expression1.png 40 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 41  Transcription is the synthesis of RNA using information in the DNA. For a protein-coding gene, the resulting RNA molecule is a faithful transcript of the gene’s protein building instructions. This type of RNA molecule is called messenger RNA (mRNA). Messenger RNA, the carrier of information from DNA to the cell’s protein-synthesizing machinery, is transcribed from the template strand of a gene. An enzyme called an RNA polymerase pries the two strands of DNA apart and joins together RNA nucleotides complementary to the DNA template strand, thus elongating the RNA polynucleotide. Suparada Khanaruksombat, Ph.D. 42  1. Ribosomal RNA (rRNA) - 85% - สร้างจาก nucluolus เพื่อไปเป็น ribosome 2. Transfer RNA (tRNA) - 10% - ให้ในการบวนการ translation โดยติดกับ anti-codon และกรดอะมิโน 3. Massenger RNA (mRNA) - 5% - ใช้เป็นแม่แบบในการสร้างโปรตีน 4. < 1% RNA อื่นๆ Suparada Khanaruksombat, Ph.D. 43  The DNA sequence where RNA polymerase attaches and initiates transcription is known as the promoter; in bacteria, the sequence that signals the end of transcription is called the terminator. Molecular biologists refer to the direction of transcription as “downstream” and the other direction as “upstream.” The stretch of DNA downstream from the promoter that is transcribed into an RNA molecule is called a transcription unit. Suparada Khanaruksombat, Ph.D. 44 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 45 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 46  Exon – ไป translation เป็นโปรตีนได้ Intron – non-coding ไม่เปลี่ยนไปเป็นโปรตีน RNA processing: addition of the 5' cap and poly-A tail. RNA splicing. Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 47  Triplets of nucleotide bases are the smallest units of uniform length that can code for all the amino acids called codon. If each arrangement of three consecutive nucleotide bases specifies an amino acid, there can be 64 (that is, 43) possible code words—more than enough to specify all the amino acids. The series of words in a gene is transcribed into a complementary series of nonoverlapping, three- nucleotide words in mRNA, which is then translated into a chain of amino acids. Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 48 Suparada Khanaruksombat, Ph.D. Mason, K. A., & Raven, P. H. (2016). Biology (11th ed.). 49  1. The beginning of an amino acid sequence is specified by a start codon located somewhere in the mRNA sequence, this is usually an AUG, but can also be a GUG. 2. The sequence of bases in a codon must follow the direction of translation. 3. The code is non-over-lapping. 4. The code is read in a fixed reading frame. 5. The end of a sequence is specified by one of three stop codons: UAA, UAG, or UGA. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). Suparada Khanaruksombat, Ph.D. 50  The message is a series of codons along an mRNA molecule, and the translator is called a transfer RNA (tRNA). The function of a tRNA is to transfer an amino acid from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome. A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long (compared to hundreds of nucleotides for most mRNA molecules). The loop extending from the other end of the L includes the anticodon, the particular nucleotide triplet that base-pairs to a specific mRNA codon. Suparada Khanaruksombat, Ph.D. 51 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 52  Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis. A ribosome consists of a large subunit and a small subunit, each made up of proteins and one or more ribosomal RNAs (rRNAs). The structure of a ribosome reflects its function of bringing mRNA together with tRNAs carrying amino acids. In addition to a binding site for mRNA, each ribosome has three binding sites for tRNA. Suparada Khanaruksombat, Ph.D. 53 Suparada Khanaruksombat,Reece, Ph.D.J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 54 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 55 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 56 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.). 57 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.).cc 58  Belk CM, Maier VB. Biology : science for life, with physiology. 4th ed. Suparada Khanaruksombat, Ph.D. 59 Suparada Khanaruksombat, Ph.D. 60 Suparada Khanaruksombat, Ph.D. Reece, J. B., Urry, L. A., & Campbell, N. A. (2016). Campbell biology (11th ed.).cc 61 Suparada Khanaruksombat, Ph.D. 62  Hoefnagels, M. l. (2018). Biology : the essentials (Third edition. ed.). Suparada Khanaruksombat, Ph.D. 63  โครงสร้างโมเลกุลของวอตสัน และคริกเป็น DNA เกลี่ยวคู่ (double helix) ถ้า DNA สายหนึ่งมี ลาดับเบสดังนี้ 5’ AACGGGTTTATGCGT 3’ ข้อใดคือลาดับเบสของ DNA ที่เป็นเบสคู่สม (complementary base) (PAT ธ.ค. 56) 1. 5’ TTGCCCAAATACGCA 3’ 2. 5’ ACGCATAAACCCGTT 3’ 3. 3’ ACGCATAAACCCGTT 5’ 4. 3’ AACGGGTTTATGCGT 5’ Suparada Khanaruksombat, Ph.D. 64  จากภาพโครงสร้างของยีนที่ประกอบด้วย 4 exon 3 intron ข้อความต่อไปนี้ถูกหรือผิด 1. ส่วน exon ที่ 1 และ 2 บางส่วน (สีเข้ม) เป็นบริเวณที่ไม่มีการแปลรหัสเป็นโปรตีน 2. start codon อยู่บน exon ที่ 1 และ stop codon อยู่บน exon ที่ 4 3. ถ้าเกิดการแทนที่เบสบริเวณสีเข้มบน exon ที่ 4 ทาให้ได้สายพอลิเพปไทด์สั้นลง 4. ภาพ mRNA ข้างบน เป็นภาพที่ยังเกิดกระบวนการ RNA splicing ไม่สมบูรณ์ Suparada Khanaruksombat, Ph.D. 65  จากลาดับเบสของยีน POSN บนดีเอ็นเอสายคู่ที่กาหนดให้ ข้อความต่อไปนี้ถูกหรือผิด 1. ยีน POSN กาหนดการสังเคราะห์ polypeptide ยาว 115 amino acid 2. ถ้าคู่เบส CG (ตัวหนา-ระบายสี) ถูกแทนที่ด้วย GC จะ ทาให้เกิด nonsense mutation และ polypeptide ที่ได้จะสั้นลง โดยหายไป 109 amino acid 3. ถ้าคู่เบส TA (ตัวขีดเส้นใต้-ระบายสี) เพิ่มขึ้น 1 ซ้า จะ ทาให้เกิด frameshift mutation และ polypeptide ที่ได้จะสั้นลง โดยหายไป 7 amino acid 4. ถ้าคู่เบส AT ที่อยู่ติดกับคู่เบส TA (ตัวขีดเส้นใต้-ระบายสี) ทางด้าน 3’ ถูกแทนที่ด้วย CG จะทาให้เกิด missense mutation Suparada Khanaruksombat, Ph.D. แต่ polypeptide ที่ได้จะมีความยาวเท่าเดิม 66